Kikuë Tachibana

Welcome to the Tachibana* lab

Welcome to the Tachibana lab website.The oocyte-to-zygote transition is the process that converts an egg, by fertilization, into a one-cell embryo that can develop into an entire organism. During the first embryonic cell cycle, egg and sperm genomes assemble into separate haploid nuclei that undergo distinct but poorly understood reprogramming mechanisms. An ongoing revolution in single-cell technologies is providing exciting new insights into zygotic reprogramming.

Fertilized germ cells must lose their „memory“ of being an egg or sperm, but how germ cell chromatin is reprogrammed after fertilization to produce a totipotent zygote is a mystery. In 2006, Yamanaka and colleagues discovered that defined factors can induce pluripotency in differentiated somatic cells. However, this process is extremely inefficient and takes days, if not weeks. In contrast, reprogramming in the zygote generates a totipotent cell within a few hours. Although factors within the egg appear to underlie zygotic reprogramming, the mechanisms remain elusive. Our current research aims to gain insights into this great and important unknown.

To gain insights into how chromatin is spatially reorganized in totipotent cells, we recently developed a chromosome conformation capture (3C)-based method called single-nucleus Hi-C (snHi-C) (Flyamer et al., Nature 2017). By extracting maternal and paternal nuclei from zygotes and separately subjecting these to snHi-C, we discovered that the two genomes have a distinct chromatin organization from each other and any other mammalian interphase genome. Moreover, the high-resolution data enabled the identification of fine chromatin features at unprecedented resolution in single cells. This method will prove invaluable to study rare cell types like adult stem cells. Having established snHi-C and described the wild-type situation, we are now in a position to combine it with functional genetics to study higher-order chromatin structure during reprogramming to totipotency.

Moreover, we have pioneered mechanistic cell biology in zygotes to study the mechanism underlying active DNA demethylation. Erasure of sperm epigenetic memory includes the loss of methylated cytosine, a repressive mark. We provided the first genetic evidence that DNA demethylation proceeds by a DNA repair-based mechanism and discovered that a surveillance mechanism monitors zygotic programming (Ladstätter & Tachibana-Konwalski, Cell 2016).

Lastly, we have a long-standing interest in the maternal age effect: as mammalian females age, chromosome missegregation in oocytes increases. Fertilization of aneuploid eggs produces aneuploid pregnancies and miscarriages. What causes the maternal age-related increase in aneuploid pregnancies has remained enigmatic for decades. A leading hypothesis is that the protein complex called cohesin deteriorates with age and this triggers chromosome missegregation. We have demonstrated that cohesin holds chromosomes together without renewal from oogenesis in fetal embryos until the meiotic division in adult females (Burkhardt et al., Curr Biol 2016). This suggests that loss of chromosomal cohesin with age is likely irreversible. We therefore next aim to understand what causes cohesin loss with age and how this process can be delayed to preserve female fertility.

Vision

Our vision is to gain molecular insights into how chromatin is reprogrammed to totipotency. Towards this overarching goal, we aim to study how chromatin is spatially reorganized during the mammalian oocyte-to-zygote transition and what determines the distinct 3D genome organization of maternal and paternal genomes within zygotes. To bring this vision to a mechanistic level, we aim to identify candidate pioneer factors responsible for natural reprogramming and gain insights into how epigenetic memory is set to a totipotent “ground state”.

Impact

An understanding of the molecular and physiological causes of the maternal age effect might have therapeutic applications in the context of in vitro fertilization and reproductive health, particularly in a society where women are delaying childbirth. Insights into the mechanisms that the zygote uses to establish totipotency might be relevant to regenerative medicine. For instance, totipotency factors may be able to reprogram proliferating cells within one cell cycle or directly reprogram differentiated cells to a totipotent state, which is earlier than currently possible.

Approach

To understand how aging impacts chromosome segregation in oocytes, we study oocytes from young and aged wild-type and genetically modified mice. The key approach is microinjection of mRNA encoding fluorescently-tagged proteins or rescue constructs into oocytes followed by 4D time-lapse microscopy to track chromosome dynamics through the meiotic divisions. We perform chromosome spreads for accurate karyotyping and quantification of chromosomal proteins.

Inside the newly formed zygote, sperm chromatin decondenses, protamines are exchanged for histones, a pronucleus is formed and chromatin remodeling erases cell-type specific epigenetic marks as part of the natural reprogramming that occurs during the first embryonic cell cycle. We are investigating factors required for these processes using a combination of mouse molecular genetics, biochemistry, TEV protease technology and live-cell microscopy.

We also established a new genome-wide approach that allows us to evaluate chromatin structure in single-cell embryos, termed high-resolution chromosome confirmation capture (Hi-C). We will use this approach to understand how chromatin structure is established in the zygote to support totipotency, and how it changes at the point of transition from totipotency to pluripotency.